Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT₁-7 cells

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT₁-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10^-12-10^-5 M) were administered to the cells. A clear dose-dependent stimulatory effect of NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10^{- 7} dose. The lowest dose of NPY that showed a significant effect was 10^{- 10} M; maximal responses were observed at 10^-6 M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10^-6 M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10^-8 M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT₁-7 cells were challenged with a Y₁ receptor agonist, (Leu³¹Pro³⁴)NPY, a Y₂ receptor agonist, NPY(13-36), or peptide YY, at doses 10^-12-10^-5 M. All four peptides stimulated LHRH release from GT₁- 7 cells with a rank-ordered potency of NPY = peptide YY > Y₁ agonist = Y₂ agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'- monophosphothioate Rp diastereomer), Ca²⁺-free DMEM and TMB-8 (3, 4, 5- trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca²⁺-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10^-7 M NPY. However, the addition of 100 μM and 250 μM TMB-8 to Ca²⁺-free DMEM almost completely blocked this NPY effect, as did 10 μM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT₁-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%. These results demonstrate for the first time that NPY directly stimulates LHRH release from superfused GT₁-7 cells in a dose-dependent manner and that this effect seems to be specific to the NPY peptide. In addition, NPY may exert its effects on LHRH neurons predominantly via receptors possessing Y₁ subtype pharmacological properties. The effects of NPY also appear to be dependent upon the mobilization of intracellular calcium from IP₃- and/or ryanodine-sensitive stores. Our findings, therefore, suggest that one mechanism by which NPY may stimulate LHRH release in vivo may be through a direct, Y₁-like receptor-mediated action on the LHRH neuron itself.